The conformation of a signal peptide bound by Escherichia coli preprotein translocase SecA

To understand the structural nature of signal sequence recognition by the preprotein translocase SecA, we have characterized the interactions of a signal peptide corresponding to a LamB signal sequence (modified to enhance aqueous solubility) with SecA by NMR methods. One-dimensional NMR studies showed that the signal peptide binds SecA with a moderately fast exchange rate (Kd approximately 10(-5) m). The line-broadening effects observed from one-dimensional and two-dimensional NMR spectra indicated that the binding mode does not equally immobilize all segments of this peptide. The positively charged arginine residues of the n-region and the hydrophobic residues of the h-region had less mobility than the polar residues of the c-region in the SecA-bound state, suggesting that this peptide has both electrostatic and hydrophobic interactions with the binding pocket of SecA. Transferred nuclear Overhauser experiments revealed that the h-region and part of the c-region of the signal peptide form an alpha-helical conformation upon binding to SecA. One side of the hydrophobic core of the helical h-region appeared to be more strongly bound in the binding pocket, whereas the extreme C terminus of the peptide was not intimately involved. These results argue that the positive charges at the n-region and the hydrophobic helical h-region are the selective features for recognition of signal sequences by SecA and that the signal peptide-binding site on SecA is not fully buried within its structure.     

The role of the membrane-spanning domain of type I signal peptidases in substrate cleavage site selection

Type I signal peptidase (SPase I) catalyzes the cleavage of the amino-terminal signal sequences from preproteins destined for cell export. Preproteins contain a signal sequence with a positively charged n-region, a hydrophobic h-region, and a neutral but polar c-region. Despite having no distinct consensus sequence other than a commonly found c-region "Ala-X-Ala" motif preceding the cleavage site, signal sequences are recognized by SPase I with high fidelity. Remarkably, other potential Ala-X-Ala sites are not cleaved within the preprotein. One hypothesis is that the source of this fidelity is due to the anchoring of both the SPase I enzyme (by way of its transmembrane segment) and the preprotein substrate (by the h-region in the signal sequence) in the membrane. This limits the enzyme-substrate interactions such that cleavage occurs at only one site. In this work we have, for the first time, successfully isolated Bacillus subtilis type I signal peptidase (SipS) and a truncated version lacking the transmembrane domain (SipS-P2). With purified full-length as well as truncated constructs of both B. subtilis and Escherichia coli (Lep) SPase I, in vitro specificity studies indicate that the transmembrane domains of either enzyme are not important determinants of in vitro cleavage fidelity, since enzyme constructs lacking them reveal no alternate site processing of pro-OmpA nuclease A substrate. In addition, experiments with mutant pro-OmpA nuclease A substrate constructs indicate that the h-region of the signal peptide is also not critical for substrate specificity. In contrast, certain mutants in the c-region of the signal peptide result in alternate site cleavage by both Lep and SipS enzymes.     

Positively charged residues, the helical conformation and the structural flexibility of the leader sequence of pALDH are important for recognition by hTom20

Tom20, a mitochondrial outer membrane receptor necessary for protein translocation, was found to interact specifically with mitochondrial preproteins. The interaction of proteins containing an N-terminal matrix targeting signal was enhanced in an hydrophobic environment and the dependence of this interaction on the alpha helical conformation of the presequence was postulated. In order to test this hypothesis and to gain insights about the features of a matrix targeting signal necessary to be recognized by the receptor machinery including Tom20, the interaction of pALDH and signal sequence mutants to Tom20 in the absence and presence of a hydrophobic environment was investigated. Here we present evidence to show that in a hydrophobic environment the interaction between Tom20 and the leader sequence is strongly dependent on the positive charges within the signal sequence as well as on the flexibility of this signal.     

Identification and characterization of receptors for protein import into chloroplasts and mitochondria

An anti-idiotypic antibody approach was used to identify chloroplast and mitochondrial protein component(s) which interact with the corresponding signal sequence. The proteins thus identified can be operationally defined as receptor(s) for import of proteins into chloroplasts and mitochondria. The import receptor(s) was found in "contact sites" between the outer and inner membrane of chloroplast envelope or of mitochondria.     

Effects of neighboring sequence environment in predicting cleavage sites of signal peptides

Signal peptide has a pivotal role in the translocation of secretory protein. Some models have been designed to predict its cleavage site. It is reported that the cleavage site has relationship with the neighboring sequence environment, i.e., hydrophobic core h-region, and the specific patterns in c-region. In some studies, this finding does facilitate the prediction of cleavage site. However, in these models, sequence environment information is merely taken account of as model inputs and no detailed investigation into its effect on the prediction of cleavage site has been made. In this work, we analyze the constraint on cleave site placed by the hydrophobic core of signal peptide and then use it to improve the performance of the signal peptide cleavage site prediction. Our model is designed as follows: firstly, a sliding window is used to scan sample and artificial neural network (ANN) is employed to give cleavage site/non-cleavage site scores. Then, based on an estimated hydrophobic h-region a correcting function is proposed to improve the prediction result, in which the sequence environment is taken into account. A trend of cleavage site is indicated by our analysis for each position, which is consistent with experimental findings. Through this correcting step, the improvement of prediction accuracy is over 7%. It therefore demonstrates the neighboring sequence environment is helpful for determination of cleavage site. Program written in Matlab can be downloaded from http://www.scucic.cn/combined model/source code.html.     

Establishment of a signal peptide with cross-species compatibility for functional antibody expression in both Escherichia coli and Chinese hamster ovary cells

Signal peptides are short peptides located at the N-terminus of secreted proteins. They characteristically have three domains; a basic region at the N-terminus (n-region), a central hydrophobic core (h-region) and a carboxy-terminal cleavage region (c-region). Although hundreds of different signal peptides have been identified, it has not been completely understood how their features enable signal peptides to influence protein expression. Antibody-derived signal peptides are often used to prepare recombinant antibodies expressed by eukaryotic cells, especially Chinese hamster ovary (CHO) cells. However, when prokaryotic Escherichia coli (E. coli) are utilized in drug discovery processes, such as for phage display selection or antibody humanization, signal peptides have been selected separately due to the differences in the expression systems between the species. In this study, we successfully established a signal peptide that enables a functional antibody to be expressed in both prokaryotic and eukaryotic cells by focusing on the importance of having an Ala residue in the c-region of the signal sequence. We found that changing Ser to Ala at only two positions significantly augmented the anti-HER2 antigen binding fragment (Fab) expression in E. coli. In addition, this altered signal peptide also retained the ability to express functional anti-HER2 antibody in CHO cells. Taken together, the present findings indicate that the signal peptide can promote functional antibody expression in both prokaryotic E. coli and eukaryotic CHO cells. This finding will contribute to the understanding of signal peptides and accelerate therapeutic antibody research.     

Competition between Sec- and TAT-dependent protein translocation in Escherichia coli.

Recently, a new protein translocation pathway, the twin-arginine translocation (TAT) pathway, has been identified in both bacteria and chloroplasts. To study the possible competition between the TAT- and the well-characterized Sec translocon-dependent pathways in Escherichia coli, we have fused the TorA TAT-targeting signal peptide to the Sec-dependent inner membrane protein leader peptidase (Lep). We find that the soluble, periplasmic P2 domain from Lep is re-routed by the TorA signal peptide into the TAT pathway. In contrast, the full-length TorA-Lep fusion protein is not re-routed into the TAT pathway, suggesting that Sec-targeting signals in Lep can override TAT-targeting information in the TorA signal peptide. We also show that the TorA signal peptide can be converted into a Sec-targeting signal peptide by increasing the hydrophobicity of its h-region. Thus, beyond the twin-arginine motif, the overall hydrophobicity of the signal peptide plays an important role in TAT versus Sec targeting. This is consistent with statistical data showing that TAT-targeting signal peptides in general have less hydrophobic h-regions than Sec-targeting signal peptides.     

Nuclear import in permeabilized protoplasts from higher plants has unique features.

The import of proteins into the nucleus is a poorly understood process that is thought to require soluble cytosolic factors in vertebrates and yeast. To test this model in plants and to identify components of the import apparatus, we developed a direct in vitro nuclear import assay by using tobacco protoplasts that were permeabilized without detergents such as digitonin or Triton X-100. Substrates were imported specifically by a mechanism that required only guanine nucleotides. Moreover, in vitro import did not require exogenous cytosol. To investigate this novel finding, we isolated a full-length cDNA encoding an Arabidopsis homolog of vertebrate and yeast nuclear localization signal receptors and produced an affinity-purified antibody. The plant receptor was tightly associated with cellular components in permeabilized protoplasts, even in the presence of 0.1% Triton X-100, indicating that this factor and probably others were retained to an extent sufficient to support import. The lectin wheat germ agglutinin bound to the nucleus; however, it did not block translocation in our system, indicating that direct interaction with polysaccharide modifications at the nuclear pore complex was probably not essential for import in plants. Other features of in vitro import included reduced but significant import at low temperature.     

Tryptophan fluorescence study on the interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with model membranes

The interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with different phospholipid vesicles was investigated by fluorescence techniques, using a synthetic mutant signal peptide in which valine at position -8 in the hydrophobic sequence was replaced by tryptophan. First it was established that this mutation in the signal sequence of prePhoE does not affect in vivo and in vitro translocation efficiency and that the biophysical properties of the synthetic mutant signal peptide are similar to those of the wild-type signal peptide. Next, fluorescence experiments were performed which showed an increase in quantum yield and a blue shift of the emission wavelength maximum upon interaction of the signal peptide with lipid vesicles, indicating that the tryptophan moiety enters a more hydrophobic environment. These changes in intrinsic fluorescence were found to be more pronounced in the presence of phosphatidylglycerol (PG) or cardiolipin (CL) than with phosphatidylcholine (PC). In addition, quenching experiments demonstrated a shielding of the tryptophan fluorescence from quenching by the aqueous quenchers iodide and acrylamide upon interaction of the signal peptide with lipid vesicles, a shielding in the case of acrylamide that was more pronounced in the presence of negatively charged lipids. Finally it was found that acyl chain brominated lipids incorporated into phospholipid bilayers were able to quench the tryptophan fluorescence of the signal peptide, with the quenching efficiency in CL vesicles being much higher than in PC vesicles. The results clearly demonstrate that the PhoE signal peptide interacts strongly with different lipid vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

The corticotropin-releasing factor receptor type 2a contains an N-terminal pseudo signal peptide

The corticotropin-releasing factor receptor type 2a (CRF(2(a)) receptor) belongs to the family of G protein-coupled receptors. The receptor possesses a putative N-terminal signal peptide that is believed to be cleaved-off after mediating the endoplasmic reticulum targeting/insertion process, like the corresponding sequence of the homologous CRF(1) receptor. Here, we have assessed the functional significance of the putative signal peptide of the CRF(2(a)) receptor and show that it is surprisingly completely incapable of mediating endoplasmic reticulum targeting, despite meeting all sequence criteria for a functional signal by prediction algorithms. Moreover, it is uncleaved and forms part of the mature receptor protein. Replacement of residue Asn(13) by hydrophobic or positively charged residues converts the sequence into a fully functional and cleaved signal peptide demonstrating that conventional signal peptide functions are inhibited by a single amino acid residue. Deletion of the domain leads to an increase in the amount of immature, intracellularly retained receptors demonstrating that the sequence has adopted a new function in receptor trafficking through the early secretory pathway. Taken together, our results identify a novel hydrophobic receptor domain in the family of the heptahelical G protein-coupled receptors and the first pseudo signal peptide of a eukaryotic membrane protein. Our data also show that the extreme N termini of the individual CRF receptor subtypes differ substantially.     

Structural determinants for signal sequence function in the mammalian endoplasmic reticulum

Signal sequences function in protein targeting to and translocation across the endoplasmic reticulum membrane. To investigate the structural requirements for signal sequence function, chimeras of the Escherichia coli LamB signal peptide and prolactin were prepared. The LamB signal peptide was chosen by virtue of the extensive biophysical and biological characterization of its activity. In vitro, nascent prolactin chains bearing the LamB signal peptide (LamB) were targeted in a signal recognition particle (SRP)-dependent manner to rough microsomes but remained protease- and salt-sensitive and translocated at low efficiency. Full translocation activity was obtained in a gain of function mutant (LamB*) in which three hydrophobic residues in the LamB hydrophobic core were converted to leucine residues. Cross-linking studies demonstrated that the LamB* signal sequence displayed markedly enhanced interactions with SRP and integral membrane proteins. In contrast, chemically denatured LamB and LamB*-precursors bound with identical efficiencies and in a salt-resistant manner to rough microsomes, suggesting that during de novo synthesis the signal sequence of LamB-bearing precursors assumes a conformation refractory to translocation. These data indicate that a leucine-rich signal sequence is necessary for optimal interaction with SRP and suggest that SRP, by maintaining the signal sequence in a conformation suitable for membrane binding, performs a chaperone function.     

Recognition of signal sequences

The hypothesis assumes that every continuous, entirely hydrophobic sequence of sufficient length, which is not involved in strong intramolecular contacts with other parts of the nascent protein chain, will function as a signal for translocation across the endoplasmic reticulum membrane or across the inner bacterial membrane. The signal peptide is proposed to be deeply immersed into a hydrophobic cleft of the receptor. Accordingly, only the entirely nonpolar peptides can be absorbed and, despite different primary structures, all of them would assume the same conformation dictated by the structure of the receptor pocket.     

Identification of hydrophobic residues in the signal sequence of mitochondrial preornithine carbamyltransferase that enhance the rate of precursor import

Previous studies employing circular dichroism and resonance energy transfer techniques have demonstrated that the signal peptide of mitochondrial preornithine carbamyltransferase (pOCT) has the potential to interact with the surface of an anionic phospholipid membrane via a short amphiphilic helical domain. Here we have used predictive secondary structure computations as a guide to localize the putative membrane binding region in the pOCT signal sequence and demonstrate that replacement of leucine residues at positions 5, 8, and 9 with the less hydrophobic residue, alanine, significantly reduces the rate of precursor import (4-5-fold compared to wild type); the amino acid substitutions had little effect, however, on the ability of a mitochondrial matrix extract to process the mutant precursor polypeptide. The mutant precursor bound to anionic liposomes with a lower affinity compared to wild-type pOCT and was inhibited to a lesser extent than pOCT during import into mitochondria in the presence of varying concentrations of liposomes. Taken together, the results suggest that this small region of the pOCT signal sequence, containing a limited number of critical hydrophobic residues, contributes to the optimal rate of precursor import, perhaps by functioning as a membrane surface-seeking entity.     

Lipid-like behavior of signal sequence peptides at air–water interface

Several protein transport processes in the cell are mediated by signal sequence peptides located at the N-terminal side of the mature protein sequence. To date, the specific interaction and the stability of these peptides at the amphipathic interface of biological membranes and the relevance of the peptide conformation when they interact with lipids is not clear. We report the surface properties and the peptide–lipid interaction of three signal sequence peptides at the air–NaCl 145 mM interface by using the Langmuir monolayer approach. These synthetic peptides have a natural sequence with a non-periodic amphiphilicity, where hydrophobic and hydrophilic residues are located on opposed sides of the peptide primary sequence. We show that signal sequence peptides form insoluble monolayers of high stability against lateral compression. At close packing, peptide molecular area, surface potential and the high stability of the peptide monolayer are indicative that signal sequence peptides are compatible with a β-sheet conformation at the interface. Structure was confirmed with PM-IRRAS and transmission FT-IR studies. The peptides show lateral miscibility with either POPC (a liquid-expanded lipid) or DPPC (a liquid-condensed lipid) in mixed peptide–lipid monolayers. This indicates that signal sequence peptides studied are laterally miscible with phospholipids independent of the phase state of the lipid.     

Inhibition of PhoE translocation across Escherichia coli inner-membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.     

Switching the sorting mode of membrane proteins from cotranslational endoplasmic reticulum targeting to posttranslational mitochondrial import

Hydrophobic membrane proteins are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, mediated by hydrophobic signal sequence. Mitochondrial membrane proteins escape this mechanism despite their hydrophobic character. We examined sorting of membrane proteins into the mitochondria, by using mitochondrial ATP-binding cassette (ABC) transporter isoform (ABC-me). In the absence of 135-residue N-terminal hydrophilic segment (N135), the membrane domain was integrated into the ER membrane in COS7 cells. Other sequences that were sufficient to import soluble protein into mitochondria could not import the membrane domain. N135 imports other membrane proteins into mitochondria. N135 prevents cotranslational targeting of the membrane domain to ER and in turn achieves posttranslational import into mitochondria. In a cell-free system, N135 suppresses targeting to the ER membranes, although it does not affect recognition of hydrophobic segments by signal recognition particle. We conclude that the N135 segment blocks the ER targeting of membrane proteins even in the absence of mitochondria and switches the sorting mode from cotranslational ER integration to posttranslational mitochondrial import.     

An antibody and anti-idiotypic antibody against the extra signal peptide of pre-aspartate aminotransferase

A peptide (extra signal peptide) comprising amino acids 1-29 of pig liver pre-mitochondrial aspartate aminotransferase (p-mAAT) was synthesized chemically. The peptide was found to block the import of rat liver p-mAAT into rat liver mitochondria. An antibody raised against the peptide immunoprecipitated rat liver p-mAAT synthesized in a rabbit reticulocyte cell-free translation system. These results suggested that the extra signal peptide sequence of p-mAAT is essential for import of p-mAAT into the mitochondria and that there is structural homology between the extra signal peptides of pig and rat liver p-mAAT. An anti-idiotypic antibody against the peptide was also prepared and purified by affinity chromatography on an Affi-Gel 10 anti-peptide IgG column and was then characterized.     

A Novel Pex14 Protein-interacting Site of Human Pex5 Is Critical for Matrix Protein Import into Peroxisomes

Protein import into peroxisomes relies on the import receptor Pex5, which recognizes proteins with a peroxisomal targeting signal 1 (PTS1) in the cytosol and directs them to a docking complex at the peroxisomal membrane. Receptor-cargo docking occurs at the membrane-associated protein Pex14. In human cells, this interaction is mediated by seven conserved diaromatic penta-peptide motifs (WXXX(F/Y) motifs) in the N-terminal half of Pex5 and the N-terminal domain of Pex14. A systematic screening of a Pex5 peptide library by ligand blot analysis revealed a novel Pex5-Pex14 interaction site of Pex5. The novel motif composes the sequence LVAEF with the evolutionarily conserved consensus sequence LVXEF. Replacement of the amino acid LVAEF sequence by alanines strongly affects matrix protein import into peroxisomes in vivo. The NMR structure of a complex of Pex5-(57–71) with the Pex14-N-terminal domain showed that the novel motif binds in a similar α-helical orientation as the WXXX(F/Y) motif but that the tryptophan pocket is now occupied by a leucine residue. Surface plasmon resonance analyses revealed 33 times faster dissociation rates for the LVXEF ligand when compared with a WXXX(F/Y) motif. Surprisingly, substitution of the novel motif with the higher affinity WXXX(F/Y) motif impairs protein import into peroxisomes. These data indicate that the distinct kinetic properties of the novel Pex14-binding site in Pex5 are important for processing of the peroxisomal targeting signal 1 receptor at the peroxisomal membrane. The novel Pex14-binding site may represent the initial tethering site of Pex5 from which the cargo-loaded receptor is further processed in a sequential manner.     

Integrin-like Protein Is Involved in the Osmotic Stress-induced Abscisic Acid Biosynthesis in Arabidopsis thaliana

We studied the perception of plant cells to osmotic stress that leads to the accumulation of abscisic acid （ABA） in stressed Arabidopsis thaliana L. cells. A significant difference was found between protoplasts and cells in terms of their responses to osmotic stress and ABA biosynthesis, implying that cell wall and/or cell wall-plasma membrane interaction are essential in identifying osmotic stress. Western blotting and immunofluorescence localization experiments, using polyclonal antibody against human integrin β1, revealed the existence of a protein similar to the integrin protein of animals in the suspension-cultured cells located in the plasma membrane fraction. Treatment with a synthetic pentapeptide, Gly-Arg-Gly-Asp-Ser （GRGDS）, which contains an RGD domain and interacts specifically with integrin protein and thus blocks the cell wall-plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, but not in protoplasts. These results demonstrate that cell wall and/or cell wall-plasma membrane interaction mediated by integrin-Iike proteins played important roles in osmotic stress-induced ABA biosynthesis in Arabidopsis thaliana.     

Activation of the PDGF β Receptor by a Persistent Artificial Signal Peptide

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF β receptor (PDGFβR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFβR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFβR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFβR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFβR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFβR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.